The invention provides a diagnostic and screening test for Alzheimer's disease (“AD”). An example of the test involves detecting abnormally enhanced phosphorylation of extracellular signal-regulated kinase type 1 or 2 (“Erk1/2”) in skin fibroblasts from AD patients after stimulating the cells with agonist such as bradykinin or other agents that stimulate the inositol 1,4,5-trisphosphate (IP3) receptor, in comparison to cells from age-matched controls. Enhanced phosphorylation may be measured by Western blot using antibodies specific for the phosphorylated protein or other similar approaches.
Accumulating evidence indicates that the early pathogenesis of Alzheimer's disease (AD) involves perturbation of intracellular calcium homeostasis and increased levels of oxidative stress that contribute to excitatory toxicity and neuronal death in the AD brain (Putney, 2000; Yoo et al., 2000; Sheehan et al., 1997). Studies have reported enhanced elevation of intracellular Ca2+ levels in AD brains as well as in peripheral cells in response to activation of bradykinin receptors and inactivation of a K+ channel (Ito et al., 1994; Etcheberrigaray et al., 1994; Hirashima, et al., 1996; Gibson et al., 1996; Etcheberrigaray et al., 1998). Critical proteins such as amyloid precursor protein (APP), presenilin 1 and presenilin 2, mutations of which are associated with the pathogenesis of AD, have been reported to induce dysregulation of both the IP3 receptor (IP3R) and the ryanodine receptor-(RYR-) mediated intracellular Ca2+ homeostasis (Yoo et al., 2000; Leissring et al., 1999; 2000; Mattson et al., 2000; Barrow et al., 2000). The alteration in cytosolic Ca2+ concentration is thought to contribute to the pathophysioloy of AD, including increased production of the neurotoxic 42 amino acid β-amyloid peptide (APβ) involved in plaque formation, hyperphosphorylation of tau protein involved in formation of neurfibrillay tangles, and enhanced general vulnerability of neurons to cell death.
Bradykinin (BK) is a potent vasoactive nonapeptide that is generated in the course of various inflammatory conditions. BK binds to and activates specific cell membrane BK receptor(s), thereby triggering a cascade of intracellular events leading to the phosphorylation of proteins known as “mitogen activated protein kinase” (MAPK; see below). Phosphorylation of proteins, the addition of a phosphate group to a Ser, Thr or Tyr residue, is mediated by a large number of enzymes known collectively as protein kinases. Phosphorylation normally modifies the function of, and usually activates, a protein. Homeostasis requires that phosphorylation be a transient process, which is reversed by phosphatase enzymes that dephosphorylate the substrate. Any aberration in phosphorylation or dephosphorylation disrupts biochemical pathways and multiple cellular functions. Such disruptions may be the basis for certain brain diseases.
Increased intracellular Ca2+ levels in response to BK is mediated at least by the “type 2” BK receptor (BKb2R), a G-protein-coupled receptor. Stimulation of BKb2R by BK activates phospholipase C (PLC) resulting in production of diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), second messengers involved in regulation of intracellular Ca2+ levels and activation of protein kinase C (PKC). The PLC/phospholipid/PKC pathway also interact with the Ras signaling pathway that activates the MAPK pathway. MAPK (or MAP kinase) refers to an enzyme family termed “mitogen activated protein kinase,” an important member of which is the “extracellular signal-regulated kinase” type 1 or 2 (“Erk1/2”) (Berridge, 1984; Bassa et al., 1999). Erk1/2 receive signals from multiple signal transductional pathways and is part of a pathway that leads to cell proliferation and differentiation by regulation of gene expression through a number of transcriptional factors including cyclic adenosine monophosphate (cAMP)-responsive element binding protein (CREB).
Erk1/2 phosphorylates tau protein at multiple Ser/Thr sites including Ser262 and Ser356 (Reynolds et al., 2000), which are in microtubule-binding regions of tau. Phosphorylation of Ser262 markedly compromises the ability of tau to assemble and stabilize microtubules (Biemat et al., 1993; Lu et al., 1993). Increased oxidative stress, aberrant expression of amyloid precursor protein (APP), and exposure to APβ cause activation of MAPK (McDonald et al., 1998; Ekinci and Shea, 1999; Grant et al., 1999) and enhanced tau phosphorylation (Greenberg et al., 1994).
Young L T et al., Neurosci Lett, 1988, 94:198-202 studied IP3 receptor binding sites in autopsied brains from 10 subjects with AD and 10 age-matched controls. In the parietal cortex and hippocampus, there was a 50-70% loss of [3H]-IP3 binding whereas no significant changes were observed in frontal, occipital and temporal cortices, caudate or amygdala. Scatchard analysis confirmed a reduction in receptor density rather than a change in affinity. Also, many neurotransmitters, hormones and growth factors act at membrane receptors to stimulate the phosphodiesterase hydrolysis of phosphatidyl-inositol 4,5-bisphosphate (PIP2) generating the comessengers IP3 and diacylglycerol (DAG). DAG stimulates PKC while IP3 was initially postulated to activate specific receptors leading to release of intracellular calcium, probably from the endoplasmic reticulum.
Though earlier reports had detected 32P-IP3 binding to liver and adrenal microsomes and to permeabilized neutrophils and liver cells, Solomon Snyder's group was the first to localize, isolate, analyze and later clone, IP3 receptors. Worley P F et al., Nature 1987;325:159-161, demonstrated high affinity, selective binding sites for 3H- and 32P-labelled IP3 in the brain at levels 100-300 times higher than those observed in peripheral tissues. These receptors were considered physiologically relevant because the potencies of various myoinositol analogues at the IP3 binding site corresponded to their potencies in releasing calcium from microsomes. Brain autoradiograms demonstrated discrete, heterogeneous localization of IP3 receptors. In 1988, this group (Supattapone S et al., J Biol Chem, 1988, 263:1530-1534, reported the solubilization, purification to homogeneity, and characterization of an IP3 receptor from rat cerebellum. The purified receptor was a globular protein that migrated in electrophoresis as one protein band with an Mr of 260 kDa. In a review, Snyder et al. (Cell Calcium, 1989, 10:337-342) noted that immunohistochemical studies with antisera to the purified receptor protein localized the receptor to a subdivision of the rough endoplasmic reticulum occurring in synaptic areas and in close association with the nuclear membrane. The IP3 receptor protein was selectively phosphorylated by cAMP-dependent protein kinase. This phosphorylation decreased 10-fold the potency of IP3 in releasing calcium from brain membranes. Ferris C D et al., Proc Natl Acad Sci USA, 1991 88:2232-2235 later studied phosphorylation of IP3 receptors with purified receptor protein reconstituted in liposomes (to remove detergent that can inhibit protein kinases). The IP3 receptor was stoichiometrically phosphorylated by protein kinase C (PKC) and CaM kinase II as well as by protein kinase A (PKA). IP3 receptors are regulated by phosphorylation catalyzed by the three enzymes which was additive and involved different peptide sequences. Phosphorylation by (1) PKC which was stimulated by Ca2+ and DAG, and (2) by CaM kinase II which required Ca2+, provided a means whereby Ca2+ and DAG, formed during inositol phospholipid turnover, regulate IP3 receptors. Chadwick C C et al., Proc Natl Acad Sci USA, 1990 87:2132-2136, described the isolation from smooth muscle of an IP3 receptor that was an oligomer of a single polypeptide with a Mr of 224 kDa. Furuichi T, et al., FEBS Lett, 1990 267:85-88 examined distribution of IP3 receptor mRNA in mouse tissues. The concentration of was greatest in cerebellar tissue. Moderate amounts of IP3 receptor mRNA were present in other brain tissue: thymus, heart, lung, liver, spleen, kidney, and uterus. Small amounts of IP3 receptor mRNA were observed in skeletal muscle and testicular tissue. Based on in situ hybridization, a considerable amount of IP3 receptor mRNA was located in smooth muscle cells, such as those of the arteries, bronchioles, oviduct and uterus. Ferris C D et al., J Biol Chem, 1992, 267:7036-7041, demonstrated serine autophosphorylation of the purified and reconstituted IP3 receptor and found serine protein kinase activity of the IP3 receptor toward a specific peptide substrate. The investigators concluded that the IP3 receptor protein and the phosphorylating activity reside in the same molecule. Ross C A et al. (Proc Natl Acad Sci USA, 1992, 89:4265-4269), cloned three IP3R cDNAs, designated IP3R-II, -III, and -IV, from a mouse placenta cDNA library. All three displayed strong homology in membrane-spanning domains M7 and M8 to the originally cloned cerebellar IP3R-I, with divergences predominantly in cytoplasmic domains. Levels of mRNA for the three additional IP3Rs in general were substantially lower than for IP3R-I, except for the gastrointestinal tract where levels were comparable. Cerebellar Purkinje cells expressed at least two and possibly three distinct IP3Rs, suggesting heterogeneity of IP3 action within a single cell. Sharp A H, Neuroscience, 1993, 53:927-42, examined in detail the distribution of IP3 receptors in the rat brain and spinal cord using immunohistochemical methods. IP3 receptors are present in neuronal cells, fibers and terminals in a wide distribution of areas throughout the CNS, including the olfactory bulb, thalamic nuclei and dorsal horn of the spinal cord, in circumventricular organs and neuroendocrine structures such as the area postrema, choroid plexus, subcommisural organ, pineal gland and pituitary. Ca2+ release mediated by the phosphoinositide second messenger system is important in control of diverse physiological processes. Studies of IP3 receptors in lymphocytes (T cells) by Snyder's group localized these receptors to the plasma membrane. Capping of the T cell receptor-CD3 complex, which is associated with signal transduction, was accompanied by capping of IP3 receptors. The IP3 receptor on T cells appears to be responsible for the entry of Ca2+ that initiates proliferative responses (Khan, A A et al., Science, 1992, 257:815-818)
Further with regard to IP3, Wilcox R A et al., Trends Pharmacol Sci, 1998, 19:467-475, noted that receptor-mediated activation of PLC to generate IP3 is a ubiquitous signalling pathway in mammalian systems. A family of three IP3 receptor subtype monomers form functional tetramers, which act as IP3 effectors, providing a ligand-gated channel that allows Ca2+ ions to move between cellular compartments. As IP3 receptors are located principally, although not exclusively, in the endoplasmic reticular membrane, IP3 is considered to be a second messenger that mobilizes Ca2+ from intracellular stores contributing to a variety of physiological and pathophysiological phenomena. Patel S et al., Cell Calcium, 1999, 25:247-264, reviewed the molecular properties of IP3 receptors. Several Ca2+-binding sites and a Ca2+-calmodulin-binding domain were mapped within the type I IP3 receptor, and studies on purified cerebellar IP3 receptors suggested a second Ca2+-independent calmodulin-binding domain. Overexpression of IP3 receptors provided further clues to the regulation of individual IP3 receptor isoforms present within cells, and the role that they play in the generation of IP3-dependent Ca2+ signals. IP3 receptors may be involved in cellular processes such as proliferation and apoptosis. Abdel-Latif AA. Exp Biol Med (Maywood) 2001 March;226(3):153-63 reviewed evidence, both from nonvascular and vascular smooth muscle, for cross talk between the cyclic nucleotides, cAMP and cGMP via their respective protein kinases, and the Ca2+-dependent- and Ca2+-independent-signaling pathways involved in agonist-induced contraction. These included the IP3-Ca2+-CaM-myosin light chain kinase (MLCK) pathway and the Ca2+-independent pathways, including PKC, MAP kinase, and Rho-kinase. Mikoshiba K et al., Sci STKE 2000 Sep. 26;2000(51):P, described the regulated release of calcium from intracellular stores by the IP3 receptor and the relationship of this release mechanism to calcium influx from the extracellular milieu through store-operated calcium channels. They disclosed a model of functional and physical coupling of intracellular and plasma membrane calcium channels.
Although AD is well known for its severe brain damage and memory loss, pathological changes are manifest elsewhere in the body and can be detected at the cellular level. Skin fibroblasts lying in the deep layer of skin reveal characteristic cellular and molecular abnormalities of AD damage. Skin fibroblasts are readily obtained and cultured for diagnostic purposes (U.S. Pat. No. 6,107,050, “Diagnostic Test for Alzheimer's Disease,” issued Aug. 22, 2000, which is incorporated herein by reference). However, there is a need for simpler, more economical, accurate and reliable methods for diagnosis of Alzheimer's disease.
It is known e.g. from U.S. Pat. No. 6,107,050, Alkon et al., that differential effects of an activator of intracellular Ca2+ release can be measured. Both healthy and Alzheimer's cell types exhibit a release of calcium from storage, but Alzheimer's cells exhibit a much greater release. Known methods for measuring the release of Ca2+(i) include fluorescent indicators, absorbance indicators or a Ca2+“patch clamp” electrode, and others, and such methods may be used for diagnostic purposes. However, there is a tremendous need for more effective techniques for measuring the differential effects of IP3R activators, for diagnostic, research, and clinical purposes.